The development of potent and selective inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid) and anandamide (an endogenous ligand for cannabinoid and vanilloid receptors), is detailed. The studies will provide not only the in vitro characterization of the inhibitors, but also their in vivo evaluation (pain, sleep, and inflammation) and characterization (PK properties, metabolism). They will clarify the role of endogenous oleamide and anandamide, establish the full scope of the utility of FAAH as a therapeutic target, and provide some of the first clinical candidates for the treatment of pain, sleep disorders, and chronic inflammatory diseases including contact dermatitis, and multiple sclerosis. Our studies have been extensive, providing the first class of selective, exceptionally potent, reversible and competitive inhibitors of FAAH and defining key structural features that impact inhibitor design. These studies not only provided a set of efficacious ?-ketoheterocycle FAAH inhibitors, but they addressed all the objectives set forth as specific aims in the prior grant period. The simultaneous potency (against FAAH) and selectivity (ABPP proteome-wide screening) optimizations provided selective inhibitors that display no significant off target activity including other potential enzyme targets, common P450 metabolizing enzymes, or hERG, and that exhibit efficacious in vivo activity in all models of chronic and neuropathic pain and inflammation. The continuation of these studies, their extensions to new classes of FAAH inhibitors, their in vitro and in vivo optimization using fundamental chemical, biochemical, and pharmacological tools, will be conducted with the intention of providing the first reversible inhibitors for examination in the clinic. In addition, studies to define the sites of action and endogenous role of every fatty acid amide signaling molecule and to prepare and utilize a screening library to annotate every uncharacterized serine hydrolase will be conducted. PUBLIC HEALTH RELEVANCE: The development and characterization of inhibitors of the enzyme fatty acid amide hydrolase (FAAH) will be pursued and could provide a new treatment for chronic and neuropathic pain. The discovery and characterization of new endogenous signaling fatty acid amides and their sites of action would provide a fundamental understanding of their physiological role and new opportunities for therapeutic intervention in a range of disorders (sleep, feeding, anxiety). The development of selective and potent inhibitors for each unannotated serine hydrolase will facilitate the delineation of their endogenous role and their evaluation as new therapeutic targets.